This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Glycosphingolipids and gangliosides participate in diverse biological processes, and their biological roles are dependent on the structures of both the oligosaccharide and the ceramide portions. We have used vibrationally cooled (VC)MALDI-FTMS for the detection of labile species followed by their efficient fragmentation by SORI-CAD and IRMPD. GM1 and GD1a gangliosides serve as trafficking receptors for cholera toxin and the related LTIIb toxin, respectively. We assume that GD1a ganglioside of human intestinal cells is not associated with lipid rafts due to its ceramide structural variation, which prevents endocytosis of the LTIIb-GD1a complex. The toxin receptors'ganglioside structures were evaluated as a moderator of this function, including ceramide chain length, level of saturation and hydroxylation, as well as glycan composition. To analyze these molecules, our previously developed method of direct coupling of TLC plates with VC-MALDI-FTMS was used. This allows direct TLC-MALDI-FTMS without adversely affecting the FT high resolution and mass accuracy by the surface irregularity of the TLC plate. Our earlier reports have described ganglioside purification from polarized intestinal epithelial cell line T-84 and monkey kidney Vero cells and functional studies on the mechanism of toxin biology. The samples were MALDI-desorbed directly off TLC plate surfaces with ~0.2 mm sampling steps. Fragmentation was subsequently performed by SORI-CAD and IRMPD. Both sialylated and highly fucosylated glycosphingolipids were observed. GC/MS analysis of released lipids and glycans were consistent with these observations. In recent experiments, GM1 has been modified with a fluorescent label and its traffic within the cells has been followed. Lencer's group has now determined that only GM1 with unsaturated acyl chains sorts efficiently from PM to the ER. Toxin binding was required, but other membrane components were also required, including cholesterol and the lipid raft protein flotillin. In the BUSM Mass Spectrometry Resource, the difference based on N-acyl fatty acid chains, degree of saturations, etc. of GM1 or fluorescent labeled GM1 is being investigated by high performance liquid chromatography coupled to an electrospray ionization (ESI) QStar Pulsar i time-of-flight (TOF) MS which combines, speed, sensitivity, high mass accuracy and high resolution. MALDI and ESI MS are also being used to verify that the fluorescent label is stable under the cellular conditions.